Scaling up in vitro transcription synthesis of RNA standards for competitive quantitative RT-PCR: looking for bigger yields.
Because the acquisition of in vitro transcription kits, for production of RNA standards, might not be affordable for some small laboratories, the current work describes some alternatives to the commercial Promega protocols. Yields of tested reactions were all higher than the ones reported for the standard Riboprobe kit (1mug RNA/mug template, 1x) and the optimized variant for high yields (5-10x). They were also as good as the ones described for the high RNA production kit RiboMAX (10-20x), exceeding them in approximately 70% of reactions. Our best results, achieved in a 500-mul format, were three to five times higher than the ones reported for the Promega kit.
Gonzalez-Perez
Idania
I
Centro de InmunoEnsayo (CIE), Immunochemistry, Calle 134 y Avenide 25 Playa, Apartado Postal 6653, CP 11600, Ciudad de la Habana, Cuba. idania_gonzalez@yahoo.es
Rosa
Iria García de la
IG
Cayarga
Anny Armas
AA
Hernández
Yenitse Perea
YP
González
Yaimé Josefina González
YJ
Víctores
Yoamna Rosario
YR
Dueñas-Carrera
Santiago
S
D016174:Hepacivirus / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D014158:Transcription, Genetic
D012367:RNA, Viral